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. 2015 Dec 21;6:10077. doi: 10.1038/ncomms10077

Figure 7. Six1 downregulates p53 via simultaneous upregulation of miR-27a and downregulation of RPL26.

Figure 7

(a) Depiction of the luciferase constructs used and the mutations made (bold) in the regions necessary for RPL26 binding and miR-27a binding to the p53 UTRs. (b) Luciferase assay performed in 293T cells transfected with the WT luciferase vector, miR-27a mimic or a scrambled mimic, and a pooled siRNA against RPL26 (siRPL26) or a control siNT (non-targeting) for 48 h. Analysis of variance (ANOVA) on mean±s.e.m. of biological triplicates for three combined experiments. (c) Western bot analysis performed on nuclear lysates and cytoplasmic lysates from MCF7 cells transiently transfected with a miR-27a mimic or a scrambled mimic and siRPL26 or siNT for 72 h. Runx1 levels were examined to demonstrate that the miR-27a mimic was functional. (d) Luciferase assay performed in 293T cells 48 h after transfection with a miR-27a or scrambled mimic and with the RPL26 WT or mutant reporter vector. ANOVA on mean±s.d. of biological triplicates for a representative experiment (of three experiments). (e) Luciferase assay performed in 293T cells transfected with the p53 WT UTR vector or with the 27a mut UTR vector and with miR-27a or scrambled mimic, and an siRPL26 pool or an siNT. ANOVA on mean±s.d. of biological triplicates for a representative experiment (of three experiments). (f) Luciferase assay performed in 293T cells 48 h after transfection with Six1 and the 3′-UTR reporter vector. ANOVA on mean±s.d. of biological triplicates for a representative experiment (of three experiments). (g) Luciferase assay performed in 293T cells 48 h after transfection with Six1 and either the WT or RPL26 mutant UTR reporter vectors. ANOVA on mean±s.d. of biological triplicates for a representative experiment (three experiments). (h) Western Blot analysis performed on nuclear lysates and cytoplasmic lysates from MCF7-Ctrl and Six1 cells transiently transfected with GFP-RPL26 or GFP. Note: When (−) is used, the correct non-targeting control was transfected in.